# US 10,768,174 B2 · Attachment of proteins to interfaces for use in nonlinear optical detection

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- Number: US 10,768,174 B2
- Document ID: US10768174B2
- Status: Granted
- Kind: B2
- Application number: 15/631,649
- Filed: 2017-06-23
- Published: 2017-12-07
- Granted: 2020-09-08
- Earliest claimed: 2014-12-23
- Assignees: Bluelight Therapeutics, Inc.
- Applicants: Bluelight Therapeutics, Inc.
- Inventors: Joshua Salafsky
- Web page: https://patentgrep.com/patent/US10768174B2

## CPC

- G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)}
- G01N 33/487: of liquid biological material
- G01N 21/63: optically excited
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Methods are disclosed for tethering a biological entity to a substrate comprising: (a) forming a supported lipid bilayer on a surface of a substrate, wherein the supported lipid bilayer comprises an anchor molecule conjugated to a first affinity tag that is present in the lipid bilayer at a concentration greater than or equal to 5 mole percent; and (b) contacting the supported lipid bilayer with a biological entity, wherein the biological entity comprises an nonlinear-active label and a second affinity tag capable of binding to the first affinity tag, thereby tethering the biological entity to the supported lipid bilayer in an oriented fashion.

## Claims

### Claim 1

A method comprising:
a) forming a supported lipid bilayer on a surface of a substrate, wherein the supported lipid bilayer comprises an anchor molecule that comprises or bears a first affinity tag that is present in the lipid bilayer at a concentration greater than or equal to 10 mole percent;
b) contacting the supported lipid bilayer with a protein at a concentration of less than 2 μM, wherein the protein comprises a second harmonic-active label, a second affinity tag capable of binding to the first affinity tag, and optionally a linker molecule positioned between the protein and the second affinity tag, thereby tethering the protein to the supported lipid bilayer in an oriented fashion while using less than 500 ng of the protein, wherein the tethered protein is tethered at a surface density of about 1×1012 molecules per cm2; and
c) detecting an optical signal arising from the tethered protein before and after contacting the tethered protein with a ligand, wherein the optical signal is second harmonic light, and wherein a change in the optical signal detected before and after contacting the tethered protein with the ligand indicates that the protein has undergone a conformational change.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first affinity and the second affinity tags are Ni-NTA and poly-histidine tags.

### Claim 3 (depends on 2)

The method of claim 2, wherein the poly-histidine tag is attached to the N-terminus of the protein.

### Claim 4 (depends on 2)

The method of claim 2, wherein the poly-histidine tag is attached to the C-terminus of the protein.

### Claim 5 (depends on 2)

The method of claim 2, wherein the poly-histidine tag comprises between 4 and 24 histidine residues.

### Claim 6 (depends on 2)

The method of claim 2, wherein the poly-histidine tag comprises 8 histidine residues.

### Claim 7 (depends on 2)

The method of claim 2, wherein the poly-histidine tag comprises 6 histidine residues.

### Claim 8 (depends on 1)

The method of claim 1, wherein the first affinity tag comprises cobalt2+-carboxylmethylaspartate (Co-CMA) and the second affinity tag comprises a poly-histidine tag.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first affinity tag comprises biotin and the second affinity tag comprises streptavidin.

### Claim 10 (depends on 1)

The method of claim 1, wherein the first affinity tag comprises biotin and the second affinity tag comprises avidin.

### Claim 11 (depends on 1)

The method of claim 1, wherein the first affinity tag comprises biotin and the second affinity tag comprises neutravidin.

### Claim 12 (depends on 1)

The method of claim 1, wherein the concentration of the anchor molecule comprising the first affinity tag is adjusted to a value ranging from 10 mole percent to 100 mole percent of the lipid bilayer.

### Claim 13 (depends on 1)

The method of claim 1, further comprising incubating the supported lipid bilayer with the protein for about 10 minutes to about 60 minutes.

### Claim 14 (depends on 1)

The method of claim 1, wherein the supported lipid bilayer comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine.

### Claim 15 (depends on 1)

The method of claim 1, wherein the anchor molecule conjugated to the first affinity tag comprises 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt).

### Claim 16 (depends on 1)

The method of claim 1, wherein the linker molecule comprises an omega-amino fatty acid or polyethylene glycol molecule.